Abnormal DNA methylation within genes of the steroidogenesis pathway two years after paediatric critical illness and association with stunted growth in height further in time

Abstract

Background: Former critically ill children show an epigenetic age deceleration 2 years after paediatric intensive care unit (PICU) admission as compared with normally developing healthy children, with stunted growth in height 2 years further in time as physical correlate. This was particularly pronounced in children who were 6 years or older at the time of critical illness. As this age roughly corresponds to the onset of adrenarche and further pubertal development, a relation with altered activation of endocrine pathways is plausible. We hypothesised that children who have been admitted to the PICU, sex- and age-dependently show long-term abnormal DNA methylation within genes involved in steroid hormone synthesis or steroid sulphation/desulphation, possibly aggravated by in-PICU glucocorticoid treatment, which may contribute to stunted growth in height further in time after critical illness.

Results: In this preplanned secondary analysis of the multicentre PEPaNIC-RCT and its follow-up, we compared the methylation status of genes involved in the biosynthesis of steroid hormones (aldosterone, cortisol and sex hormones) and steroid sulphation/desulphation in buccal mucosa DNA (Infinium HumanMethylation EPIC BeadChip) from former PICU patients at 2-year follow-up (n = 818) and healthy children with comparable sex and age (n = 392). Adjusting for technical variation and baseline risk factors and corrected for multiple testing (false discovery rate < 0.05), former PICU patients showed abnormal DNA methylation of 23 CpG sites (within CYP11A1, POR, CYB5A, HSD17B1, HSD17B2, HSD17B3, HSD17B6, HSD17B10, HSD17B12, CYP19A1, CYP21A2, and CYP11B2) and 4 DNA regions (within HSD17B2, HSD17B8, and HSD17B10) that were mostly hypomethylated. These abnormalities were partially sex- (1 CpG site) or age-dependent (7 CpG sites) and affected by glucocorticoid treatment (3 CpG sites). Finally, multivariable linear models identified robust associations of abnormal methylation of steroidogenic genes with shorter height further in time, at 4-year follow-up.

Conclusions: Children who have been critically ill show abnormal methylation within steroidogenic genes 2 years after PICU admission, which explained part of the stunted growth in height at 4-year follow-up. The abnormalities in DNA methylation may point to a long-term disturbance in the balance between active sex steroids and mineralocorticoids/glucocorticoids after paediatric critical illness, which requires further investigation.

Keywords: Aldosterone; Children; Cortisol; Critical illness; DNA methylation; Growth; Height; Paediatrics; Sex steroids; Steroidogenesis.

Fig. 1

Diagram of study participants. PICU: paediatric intensive care unit, PEPaNIC: Paediatric Early versus Late Parenteral Nutrition in Intensive Care Unit, RCT: randomised controlled trial

Fig. 2

Schematic overview situating the studied genes within the different branches of the steroidogenesis pathway. CYP11A1 encodes the first and rate-limiting enzyme in the steroidogenic pathway. FDX1, FDX2 and FDXR encode ferredoxin (also called adrenodoxin) and ferredoxin reductase which are involved in electron transfer to CYP11A1. HSD3B1 and HSD3B2 encode the 3β-hydroxysteroid dehydrogenases that are involved in the synthesis of sex steroids, aldosterone and cortisol. CYP17A1 encodes the enzyme catalysing the next step towards sex steroid production and POR, CYB5A and CYB5B encoding P450 oxidoreductase and cytochrome b5 are needed as mediators of electron transport to CYP17A1 for its 17,20-lyase activity. HSD17B1, HSD17B2, HSD17B3, HSD17B6, HSD17B7, HSD17B8, HSD17B10, HSD17B11, HSD17B12 and HSD17B14 encode several 17β-hydroxysteroid dehydrogenases and are a group of enzymes that mainly catalyse the interconversion of dehydroepiandrosterone <—> androstenediol, androstenedione <—> testosterone, and estrone <—> estradiol, with enzyme-specific substrate specificity/preferential direction of the reaction (see Additional File 1 Table A1). CYP19A1 encodes aromatase which is responsible for the conversion of male to female sex hormones. CYP21A2 encodes 21-hydroxylase and CYP11B1 encodes 11β-hydroxylase that are required for the synthesis of both aldosterone and cortisol. CYP11B2 encodes 11β-hydroxylase which is committed to the final steps of aldosterone synthesis. SULT2A1, SULT2B1, SULT1E1 encode enzymes responsible for sulphation of steroids and STS encodes steroid sulphatase

Fig. 3

Methylation status of differentially methylated positions in former PICU patients as compared with healthy children. The boxplots show a univariate presentation of the methylation status (β-value) of the CpG sites that were differentially methylated between former PICU patients (red) and matched healthy children (blue). The CpG sites are grouped per gene and positioned based on their location within the gene. The central lines of the boxplots depict the medians, the boxes the interquartile ranges, and the whiskers are drawn to the furthest point within 1.5 times the interquartile range from the box. PICU: paediatric intensive care unit, UTR: untranslated region

Fig. 4

Methylation status of CpG sites within regions differentially methylated between former PICU patients and healthy children. The boxplots show a univariate presentation of the methylation status (β-value) of the CpG sites located within the regions of the HSD17B2, HSD17B8 and HSD17B10 genes identified as differentially methylated between former PICU patients (red) and matched healthy children (blue). The CpG sites are grouped per gene and positioned based on their location within the gene. The central lines of the boxplots depict the medians, the boxes the interquartile ranges, and the whiskers are drawn to the furthest point within 1.5 times the interquartile range from the box. PICU: paediatric intensive care unit

Fig. 5

Interaction with sex or age of abnormal DNA methylation in former PICU patients vs healthy children. The boxplots show a univariate presentation of the methylation status (β-value) of the CpG sites for which, adjusting for baseline risk factors and technical variation, a significant interaction was found between differential methylation in former PICU patients (red) vs matched healthy children (blue) and sex (panel A) or “age at exposure” (panel B). The central lines of the boxplots depict the medians, the boxes the interquartile ranges, and the whiskers are drawn to the furthest point within 1.5 times the interquartile range from the box. PICU: paediatric intensive care unit

Fig. 6

Differentially methylated CpG sites between former PICU patients who received glucocorticoids during their stay in the PICU versus those who did not. The boxplots show a univariate presentation of the methylation status (β-value) of the CpG sites for which, adjusting for baseline risk factors and technical variation, a significant difference was found between methylation status in former PICU patients who received glucocorticoid treatment in the PICU (orange) vs those who did not (green). The central lines of the boxplots depict the medians, the boxes the interquartile ranges, and the whiskers are drawn to the furthest point within 1.5 times the interquartile range from the box. PICU: paediatric intensive care unit